We have continued our efforts in the development of high speed liquid chromatography for the separation of biochemically meaningful mixtures. First, we have introduced high performance ion pair partition chromatography for the rapid separation of hydrophilic substances, e.g. catecholamines, thyroid hormones, amino acids. In this method, small particle diameter porous silica is coated with an aqueous stationary phase at a sufficient pH that the solutes will be ionized. In addition the phase contains a salt with an appropriate counterion. Separation results mainly from differences in ion pair solubility in the organic mobile phase. Remarkable rapid separations are possible. Secondly, we have applied high performance liquid chromatography to the separation of phenylthio-hydantoin derivatives of amino acids, an important analysis problem in protein sequencing by the Edman procedure. Eighteen PTH- amino acids can be resolved in under 40 minutes using gradient elution and adsorption chromatography. Finally, we have established the principles of optimization of liquid chromatography for trace analysis. Detection limits can frequently be improved by 100 times or greater simply by increasing the sample volume injected.